Genetic variation versus recombination rate in a structured population of mice

The correlation between genetic variation and recombination rate was investigated in a structured mouse population. Nucleotide sequence data from 19 autosomal DNA loci from eight inbred strains of mouse (Mus musculus) sampled from three major subspecies were analyzed. The recombination rate was estimated from the comparison of genetic and physical map distances between markers flanking a 10-cM region of each locus. The strains were categorized into four groups (subpopulations) based on geography. By partitioning the genetic diversity into within-group and among-group variation, we detected a positive correlation between the recombination rate and nucleotide diversity within groups. The level of nucleotide differentiation among groups (G(ST)) showed a negative correlation with the rate of recombination. There was no significant correlation between recombination rate and nucleotide diversity when data from different subpopulations were pooled. No correlation was detected between recombination rate and nucleotide divergence of M. musculus and M. spicilegus. These patterns deviate from the strict neutral expectation under the constant nucleotide substitution rate, and they are likely to have been formed either by a hitchhiking effect of positively selected mutants or by background selection of deleterious mutants occurring in a subdivided population. Our series of comparisons show that because a real population always has some structure, incorporation of its information is important in detecting non-neutral evolution.     

The Evolutionary Advantage of Recombination

The controversy over the evolutionary advantage of recombination initially discovered by Fisher and by Muller is reviewed. Those authors whose models had finite-population effects found an advantage of recombination, and those whose models had infinite populations found none. The advantage of recombination is that it breaks down random linkage disequilibrium generated by genetic drift. Hill and Robertson found that the average effect of this randomly-generated linkage disequilibrium was to cause linked loci to interfere with each other's response to selection, even where there was no gene interaction between the loci. This effect is shown to be identical to the original argument of Fisher and Muller. It also predicts the "ratchet mechanism" discovered by Muller, who pointed out that deleterious mutants would more readily increase in a population without recombination. Computer simulations of substitution of favorable mutants and of the long-term increase of deleterious mutants verified the essential correctness of the original Fisher-Muller argument and the reality of the Muller ratchet mechanism. It is argued that these constitute an intrinsic advantage of recombination capable of accounting for its persistence in the face of selection for tighter linkage between interacting polymorphisms, and possibly capable of accounting for its origin.     

Genetic studies of coliphage P1. II. Relatedness to P7.

Using semiquantitative spot tests, 107 independently isolated amber mutants of P1 were shown to be rescued by a nonpermissive strain of Escherichia coli lysogenic for P7 (previously called phiamp), indicating extensive genetic relatedness between P1 and P7. The amount of rescue observed varied with mutants from different genetic linkage clusters of P1. Although these rescue tests cannot distinguish between recombination, complementation, transactivation, or combinations thereof, a major role is indicated for recombination.     

Genetic studies of coliphage P1. III. Extended genetic map.

Using semiquantitative spot tests, 107 independently isolated amber mutants of P1 were shown to be rescued by a nonpermissive strain of Escherichia coli lysogenic for P7 (previously called phiamp), indicating extensive genetic relatedness between P1 and P7. The amount of rescue observed varied with mutants from different genetic linkage clusters of P1. Although these rescue tests cannot distinguish between recombination, complementation, transactivation, or combinations thereof, a major role is indicated for recombination.     

Genetic Recombination in Crosses Between Streptomyces aureofaciens and Streptomyces rimosus

Polsinelli, M. (University of Pavia, Pavia, Italy), and Maria Beretta. Genetic recombination in crosses between Streptomyces aureofaciens and Streptomyces rimosus. J. Bacteriol. 91:63-68. 1966.-Biochemical mutants were obtained from Streptomyces rimosus and S. aureofaciens by ultraviolet irradiation. Crosses were performed between auxotrophic strains of S. rimosus and S. aureofaciens with positive results. Data are reported which indicate that the interaction observed in some crosses is due to gene recombination.     

Fixation probability in a two-locus model by the ancestral recombination-selection graph

We use the ancestral influence graph (AIG) for a two-locus, two-allele selection model in the limit of a large population size to obtain an analytic approximation for the probability of ultimate fixation of a single mutant allele A. We assume that this new mutant is introduced at a given locus into a finite population in which a previous mutant allele B is already segregating with a wild type at another linked locus. We deduce that the fixation probability increases as the recombination rate increases if allele A is either in positive epistatic interaction with B and allele B is beneficial or in no epistatic interaction with B and then allele A itself is beneficial. This holds at least as long as the recombination fraction and the selection intensity are small enough and the population size is large enough. In particular this confirms the Hill-Robertson effect, which predicts that recombination renders more likely the ultimate fixation of beneficial mutants at different loci in a population in the presence of random genetic drift even in the absence of epistasis. More importantly, we show that this is true from weak negative epistasis to positive epistasis, at least under weak selection. In the case of deleterious mutants, the fixation probability decreases as the recombination rate increases. This supports Muller's ratchet mechanism to explain the accumulation of deleterious mutants in a population lacking recombination.     

Mitotic cyclins regulate telomeric recombination in telomerase-deficient yeast cells

Telomerase-deficient mutants of Saccharomyces cerevisiae can survive death by senescence by using one of two homologous recombination pathways. The Rad51 pathway amplifies the subtelomeric Y' sequences, while the Rad50 pathway amplifies the telomeric TG(1-3) repeats. Here we show that telomerase-negative cells require Clb2 (the major B-type cyclin in this organism), in association with Cdc28 (Cdk1), to generate postsenescence survivors at a normal rate. The Rad50 pathway was more sensitive to the absence of Clb2 than the Rad51 pathway. We also report that telomerase RAD50 RAD51 triple mutants still generated postsenescence survivors. This novel Rad50- and Rad51-independent pathway of telomeric recombination also appeared to be controlled by Clb2. In telomerase-positive cells, a synthetic growth defect between mutations in CLB2 and RAD50 or in its partners in the conserved MRX complex, MRE11 and XRS2, was observed. This genetic interaction was independent of Mre11 nuclease activity but was dependent on a DNA repair function. The present data reveal an unexpected role of Cdc28/Clb2 in telomeric recombination during telomerase-independent maintenance of telomeres. They also uncover a functional interaction between Cdc28/Clb2 and MRX during the control of the mitotic cell cycle.     

Spheroplast fusion as a mode of genetic recombination in mycobacteria

Spheroplasts were prepared from two carotenoid pigment mutants of Mycobacterium aurum named NgR9 and A11, which were obtained by the chemical mutagenesis of the wild type strain A+ with N-methyl-N'-nitro-N-nitrosoguanidine. The carotenoid pigments and the alpha- and beta-mycolic acids were taken as genetic markers and the recombinants were selected on the basis of their colour on L?wenstein-Jensen medium. Spheroplasts of the two mutants were mixed in a 1:1 ratio and were treated with 40% (w/v) polyethylene glycol 6000 for 5 min at 37 degrees C. The frequency of NgR9 X A11 recombination in optimal conditions was about 2.5 X 10(-3). The recombinants selected on the basis of their carotenoid pigment profile were also tested for their alpha- and beta-mycolic acids as a second genetic marker. The results were further confirmed by electron microscopy. The optimal conditions for spheroplast fusion as a mode of genetic recombination in M. aurum are described.     

Genetic analysis of the recG locus of Escherichia coli K-12 and of its role in recombination and DNA repair.

We describe a transposon insertion that reduces the efficiency of homologous recombination and DNA repair in Escherichia coli. The insertion, rec-258, was located between pyrE and dgo at min 82.1 on the current linkage map. On the basis of linkage to pyrE and complementation studies with the cloned rec+ gene, rec-258 was identified as an allele of the recG locus first reported by Storm et al. (P. K. Storm, W. P. M. Hoekstra, P. G. De Haan, and C. Verhoef, Mutat. Res. 13:9-17, 1971). The recG258 mutation confers sensitivity to mitomycin C and UV light and a 3- to 10-fold deficiency in conjugational recombination in wild-type, recB recC sbcA, and recB recC sbcB sbcC genetic backgrounds. It does not appear to affect plasmid recombination in the wild-type. A recG258 single mutant is also sensitive to ionizing radiation. The SOS response is induced normally, although the basal level of expression is elevated two- to threefold. Further genetic studies revealed that recB recG and recG recJ double mutants are much more sensitive to UV light than the respective single mutants in each case. However, no synergistic interactions were discovered between recG258 and mutations in recF, recN, or recQ. It is concluded that recG does not fall within any of the accepted groups of genes that affect recombination and DNA repair.     

Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination

The recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis , a non-pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA ⁻ strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA ⁻ mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA ⁻ mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.     

Genetic recombination between alpha-toxin mutants of Staphylococcus aureus

McClatchy, J. K. (The University of Texas Southwestern Medical School, Dallas), and E. D. Rosenblum. Genetic recombination between alpha-toxin mutants of Staphylococcus aureus. J. Bacteriol. 92:580-583. 1966.-A demonstration of genetic recombination between Staphylococcus aureus nonhemolytic mutants was attempted by means of transduction. The results of two-point reciprocal transductions placed the mutants into two genetic groups. Recombination within each group was not detectable within the limits of the method, but hemolytic recombinants were obtained in transductional crosses when donor and recipient were from different groups. At least two genetic loci are therefore involved in alpha-toxin production. The 11 mutants of group II were fibrinolysin-negative. The recombinants were always found to be restored to fibrinolysin production as well as to alpha-toxin production. These data suggest the existence of a pleiotropic gene simultaneously affecting the synthesis of both alpha toxin and fibrinolysin. The nine mutants of group I were fibrinolysin-positive. Group I members are postulated to be alpha-toxin structural mutants. Three mutants were also negative for bound coagulase, but no linkage was observed between the locus controlling bound coagulase and the loci for either fibrinolysin or alpha-toxin production.     

New types of Escherichia coli K-12 facultative recombination deficient mutants resistant to ultraviolet.

Numerous facultative temperature sensitive recombination deficient mutants of Escherichia coli K-12 strain 108 were isolated after mutagenization with nitrosoguanidine. The majority of the mutants were resistant to UV irradiation. Three mutants, KBP72, KBP169 and KBP610, with marked recombination deificiency (300 to 15,000 times) at 42 degrees C, were UV resistant; their sensitivity to mitomycin C was altered only slightly or not at all. Mutation KBP72 was co-transduced with ilv (83 unit on E. coli genetic map). The mutant is not able to form a functional recombinat structure. Two other mutations are located between 0 and 19 unit of the genetic map.     

Temperature-Sensitive Mutants of Murine Leukemia Virus IV. Further Physiological Characterization and Evidence for Genetic Recombination

Several temperature-sensitive mutants of the Rauscher strain of murine leukemia virus representing three distinct physiological groups have been further characterized. Genetic recombination between different pairs of these mutants has been demonstrated. Several representative genetic recombinants were isolated and shown to replicate equally well at the permissive (31 C) and nonpermissive (38 C) temperatures and to have serological characteristics of the wild-type parental virus. Alternative models for the mechanisms involved in recombination between type C RNA viruses are discussed.     

The interplay of RecA-related proteins and the MND1-HOP2 complex during meiosis in Arabidopsis thaliana

During meiosis, homologous chromosomes recognize each other, align, and exchange genetic information. This process requires the action of RecA-related proteins Rad51 and Dmc1 to catalyze DNA strand exchanges. The Mnd1-Hop2 complex has been shown to assist in Dmc1-dependent processes. Furthermore, higher eukaryotes possess additional RecA-related proteins, like XRCC3, which are involved in meiotic recombination. However, little is known about the functional interplay between these proteins during meiosis. We investigated the functional relationship between AtMND1, AtDMC1, AtRAD51, and AtXRCC3 during meiosis in Arabidopsis thaliana. We demonstrate the localization of AtMND1 to meiotic chromosomes, even in the absence of recombination, and show that AtMND1 loading depends exclusively on AHP2, the Arabidopsis Hop2 homolog. We provide evidence of genetic interaction between AtMND1, AtDMC1, AtRAD51, and AtXRCC3. In vitro assays suggest that this functional link is due to direct interaction of the AtMND1-AHP2 complex with AtRAD51 and AtDMC1. We show that AtDMC1 foci accumulate in the Atmnd1 mutant, but are reduced in number in Atrad51 and Atxrcc3 mutants. This study provides the first insights into the functional differences of AtRAD51 and AtXRCC3 during meiosis, demonstrating that AtXRCC3 is dispensable for AtDMC1 focus formation in an Atmnd1 mutant background, whereas AtRAD51 is not. These results clarify the functional interactions between key players in the strand exchange processes during meiotic recombination. Furthermore, they highlight a direct interaction between MND1 and RAD51 and show a functional divergence between RAD51 and XRCC3.     

Gin mutants that can be suppressed by a Fis-independent mutation.

The Gin invertase of bacteriophage Mu mediates recombination between two inverted gix sites. Recombination requires the presence of a second protein, Fis, which binds to an enhancer sequence. We have isolated 24 different mutants of Gin that are impaired in DNA inversion but proficient in DNA binding. Six of these mutants could be suppressed for inversion by introduction of a second mutation, which when present in the wild-type gin gene causes a Fis-independent phenotype. Only one of the six resulting double mutants shows an inversion efficiency which is comparable to that of the wild-type Gin and which is independent of Fis. The corresponding mutation, M to I at position 108 (M108I), is located in a putative alpha-helical structure, which in the homologous gamma delta resolvase has been implicated in dimerization. The properties of the M108I mutant suggest that in Gin this dimerization helix might also be the target for Fis interaction. The five other mutants that show a restored inversion after introduction of a Fis-independent mutation appear to be completely dependent on Fis for this inversion. The corresponding mutations are located in different domains of the protein. The properties of these mutants in connection with the role of Fis in inversion will be discussed.     

Spectroscopic and redox properties of sym1 and (M)F195H: Rhodobacter capsulatus reaction center symmetry mutants which affect the initial electron donor.

The redox properties, absorption, electroabsorption, CD, EPR, and P+QA- recombination kinetics have been measured for the special pairs of two mutants of Rhodobacter capsulatus reaction centers involving amino acid changes in the vicinity of the special pair, P. Both mutants symmetrize amino acid residues so that portions of the M-sequence are replaced with L-sequence: sym1 symmetrizes all residues between M187 and M203, whereas (M)F195H is a single amino acid subset of the sym1 mutation. (M)F195H introduces a His residue in a position where it is likely to form a hydrogen bond to the acetyl group of the M-side bacteriochlorophyll of P. For both mutants compared with wild-type, (i) the redox potential is at least 100 meV greater, (ii) the P+QA- recombination rate is about twice as fast at room temperature, and (iii) the large electroabsorption feature for the QY band of P is shifted relative to the absorption spectrum. The comparison of the properties observed for the sym1 and (M)F195H reaction center mutants and the differences between these mutants and wild-type suggest that residue M195 is an important determinant of the properties of the special pair.     

ruvA and ruvB mutants specifically impaired for replication fork reversal

Replication fork reversal (RFR) is a reaction that takes place in Escherichia coli at replication forks arrested by the inactivation of a replication protein. Fork reversal involves the annealing of the leading and lagging strand ends; it results in the formation of a Holliday junction adjacent to DNA double-strand end, both of which are processed by recombination enzymes. In several replication mutants, replication fork reversal is catalysed by the RuvAB complex, originally characterized for its role in the last steps of homologous recombination, branch migration and resolution of Holliday junctions. We present here the isolation and characterization of ruvA and ruvB single mutants that are impaired for RFR at forks arrested by the inactivation of polymerase III, while they remain capable of homologous recombination. The positions of the mutations in the proteins and the genetic properties of the mutants suggest that the mutations affect DNA binding, RuvA-RuvB interaction and/or RuvB-helicase activity. These results show that a partial RuvA or RuvB defect affects primarily RFR, implying that RFR is a more demanding reaction than Holliday junction resolution.     

Hin recombinase mutants functionally disrupted in interactions with Fis

A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649-1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.     

Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.